Figure 6. Direct interaction between normal C1INH and C1INH_{Gly162_Pro206del}.

(A) Colocalization of normal and mutated C1INH. Widefield microscopy of HeLa cells cultured for 48 hours after cotransfection with SERPING1[WT]-mCherry and SERPING1[WT]-HA or SERPING1[c.551_685del]-HA. C1INH-HA was visualized with HA-specific antibody (green) and nuclei with Hoechst (blue). Scale bars: 10 μm. (B and C) Western blot analysis of total protein in medium as well as soluble and insoluble protein fractions derived from HeLa cells cultured for 72 hours after transfection or cotransfection with 900 ng plasmid DNA in total. Separation of soluble and insoluble C1INH fractions by centrifugation of whole cell lysate at 12,000g for 20 minutes at 4°C. (B) Detection of C1INH_{Gly162_Pro206del} in the insoluble fraction. HeLa cells were transfected with 900 ng pSERPING1[WT] or pSERPING1[c.551_685del] or cotransfected with 450 ng pSERPING1[WT] and 450 ng pSERPING1[c.551_685de]. C1INH levels were detected using PAb C1INH antibody. (C) Normal C1INH in the insoluble fraction induced by C1INH_{Gly162_Pro206del}. HeLa cells were cotransfected with 450 ng pSERPING1[WT]-HA and either 450 ng pcDNA or pSERPING1[c.551_685del]. Normal C1INH-HA levels were detected with HA-specific antibody. (D) Direct protein-protein interaction between normal and mutated C1INH. Coimmunoprecipitation on whole cell lysate from HeLa cells cultured for 48 hours after transfection or cotransfection with 40 μg plasmid DNA in total. HeLa cells were transfected with pSERPING1[WT]-V5, pSERPING1[WT]-HA, or pSERPING1[c.551_685del]-HA, or cotransfected with pSERPING1[WT]-V5 and pSERPING1[WT]-HA or pSERPING1[c.551_685del]-HA. A small amount of the whole cell lysate was saved (input) and the remaining lysate incubated with anti-V5 coupled beads to capture normal V5-tagged C1INH and interacting proteins (co-IP). Presence of normal C1INH-V5 and C1INH[Variant]-HA was visualized with V5- and HA-specific antibodies as relevant. (A) Data are representative of findings from more than 3 biological replicates. (A–C) Similar results were seen in at least 2 independent experiments. (B–D) Transfections were carried out in triplicate (n = 3).